Telomerase is a special reverse transcriptase, which is a ribonucleoprotein (RNP) complex formed of RNA in association with a protein, and comprises 3 main components: a telomerase RNA (TERC) template, a telomerase catalytic subunit (Telomerase Reverse Transcriptase, TERT), and a telomerase-associated protein (TLP). TERC is a template for use in an extension reaction by the telomerase, which has about 450 bases including a template sequence 5′-CUAACCCUAAC-3′ (SEQ ID NO: 6). TERT is a catalytic subunit of the telomerase, and TLP is the regulatory unit of the telomerase. Telomerase mainly functions to synthesize and extend the telomere-TTAGGG-repeat sequence (G sequence) by using a 3′ terminus of the telomeric DNA at the end of the chromosome as a primer and using its own RNA as a template, so as to compensate the telomeric DNA sequence lost during cell division. In some particular tissue cells, for example, germ cells, embryonic stem cells, hematopoietic stem cells, peripheral blood lymphocytes, hair, skin, and endometrial cells, and other highly dividing tissue cells, activated telomerase is expressed at a low level. However, no telomerase activity is detected in a normal mature somatic cell, and aging and death of the cells are caused by the gradually shortening of the telomere. Very few somatic cells may escape from the programmed aging by accidently activating the telomerase. However, the prolonged survival of these cells may provide opportunities for build-up of additional genetic damages, causing progressive tumor development, that is, cancerization. Therefore, telomerase reactivation is a critical step in the transition of somatic cells to tumor cells, i.e., cancerization. Until now, it is found that a majority of the malignant tumor cells are telomerase positive (>85%), and the telomerase positive rate in the peri-tumorous and normal tissues are very low (<5%). Accordingly, telomerase is a generally recognized tumor marker of high specificity. It is anticipated that the detection of telomerase may be developed into a powerful tool in the detection and molecular diagnostics of cancers. The specimen for telomerase detection may be derived from cultured cells, surgically removed tissues, needle aspiration biopsies, hydrothorax, ascites, bladder or pancreatic duct washings, secretions taken by cotton swabs, sputum, and urine.
The existing methods for detecting the telomerase activity include: 1. non-PCR based detection methods, wherein the product resulted from the telomerase catalyzed extension reaction is directly analyzed and determined by isotope, fluorescent, and chemiluminescent assay. Due to the limited sensitivity, they are substantially replaced by the detection methods based on PCR amplification at present; 2. conventional PCR-based detection methods of the telomerase activity, including Telomeric Repeats Amplification Protocols (TRAPs) and methods derived therefrom, in which the amplification products are a series of DNA fragments of different lengths; and 3. non-TRAP PCR-based detection methods, including Premature Termination of telomere extension-PCR (PTEP) and Anchored-Extension and Telomeric Complements Amplification (AETCA), in which the amplification product is a specific DNA fragment of a fixed length.
The conventional TRAP method requires the troublesome polyacrylamide gel electrophoresis and staining steps. Later to this, improvements have been made by many researchers, in particular, the introduction of internal standard enables the accurate semi-quantitative determination with the aid of some analytical instruments. However, the improvements are made only with respect to the detection means post PCR, the core TRAP-PCR reaction used is unchanged, and thus the following inherent defects of the TRAP method cannot be overcome:
(1) The amplification efficiency is low. Because in the TRAP method, the product resulting from the telomerase catalyzed extension reaction is directly used as a template for PCR, and the obtained PCR products are a series of lanes that are from dozens of by to hundreds of by in length. The uncertainty of the amplification products causes great limitation to the amplification efficiency, thereby reducing the sensitivity.
(2) A specificity-related problem exists. The uncertainty of the TRAP amplification products leads to a problem that the analysis of the results trends to be interfered by non-specific PCR amplification.
(3) The repeatability/stability is poor. Because for the TRAP method, a cell lysate is directly added into the PCR system and the cell lysate generally contains numerous ingredients inhibiting the PCR reaction, the failure of the entire detection may generally be caused.
(4) Limitations on the sensitivity are caused by the system. The cell lysate may be added to the detection system in a volume that is not larger than 4% of the total volume, which limits the detection sensitivity.
(5) The applicability is limited. The method is not applicable to the detection of the inhibition effect on telomerase, since PCR is linked to the telomerase catalyzed extension reaction, and the telomerase inhibitor has a strong inhibition on PCR.
(6) The real-time fluorescent quantitative PCR is difficult to be performed. Due to the interference from the non-specific PCR amplification, SYBR staining is not applicable, and the TaqMan probe method is not applicable since the probe region is overlapped with the downstream primer region.
The PTEP method was reported by CHEN Ran, et al in 2003 and a patent (ZL00127583.6) has been granted thereto in China in the same year. The principle of the method is that the PCR primer plays a function by complementary binding of its 3′ end to a template, and the mismatch in a middle part of the chain does not hinder the initiation of the PCR reaction. In this method, a specially constructed 159 bp DNA is introduced, and the telomerase primer TS is completely complementary to two ends of the 159 bp DNA except for two bases at the 3′ end. By adding no dTTP, the telomerase catalyzed extension reaction is restrictedly terminated in a unit, to obtain an early terminated extension product TS+AGGG, after that, dTTP and a Taq enzyme are added to the system, and the TS+AGGG is used as a starting primer to initiate the amplification of the 159 bp DNA. After 2 PCR cycles, because the completely complementary sequence of TS is integrated into a new product, TS is used as a PCR primer for amplifying the new product. The amplification product is a specific fragment of the 159 bp DNA, and can be appropriately assayed by various conventional analysis methods. The main improvement of the method over the TRAP method is that in contrast to the uncertain amplification products of varying sizes in the TRAP method, the amplification product is changed to a specific amplified fragment. However, because the telomerase catalyzed extension reaction is linked to PCR and no improvements are made with respect to the inhibition of the cell lysate on PCR, the repeatability/stability is not good enough. Moreover, the process is troublesome because the reagents (dTTP and the Taq enzyme) need to be supplemented tubewise during the reaction.
The AETCA method was invented by CHEN Ran et al in 2012. In the method, an anchored telomerase primer is used for extension of the telomeric G sequence, and another template probe having a pair of universal PCR primer sequences and 6 units of telomeric C complementary sequences is hybridized and bound to the anchored telomeric G sequence. The unbound template probe is removed by repeatedly washing, and the template probe bound to the anchored telomeric G sequence is amplified in subsequent PCR reaction. The product is a specific DNA fragment of a fixed length. In the method, the PCR inhibiting materials may be removed by the repeated washing steps, and the template probe is bound to the anchored telomeric G sequence as multiple copies, such that the PCR template is enriched, and the overall sensitivity of the method is improved. However, the repeated washing steps are troublesome, and the binding of the template probe to the target G sequence has a high requirement on temperature control and is influenced by the variation in ambient temperature. Where the room temperature is too low, non-specific binding may occur, leading to false positive results and thus causing the fluctuation and inconsistency of the results. In the present invention, considerable improvements are made on the AETCA method by removing the template probe unbound to the G sequence by introducing an inhibitory probe mediated enzymatic cleavage reaction to replace the washing steps.